Traumatic stress, such as from physical trauma, open-heart surgery or severe burns, is associated with secondary complications leading to morbidity and death in subjects that receive the traumatic insult. The mechanisms by which such serious consequences develop in a subset of the population are incompletely understood but are believed to involve neuroimmunological processes that may, for example, result in altered responses to infection, leading to sepsis and organ failure. In the intensive care unit (ICU) trauma and sepsis are leading causes of mortality. The rate of this type of ICU mortality has remained largely unchanged for several decades and new approaches for early detection of at-risk individuals and effective interventions that reduce the immunosuppressive effects of stress, organ failure and death are desperately needed.
A neuroimmunological stress response (NSR) caused by a traumatic, xenobiotic, oxidative, nociceptive or physiological stressor is often characterized by the release of peptides such as Substance P or calcitonin-related peptide (CGRP) from sensory nerves into the tissues and the triggering of complex downstream events such as acute inflammation and apoptotic cell death (in the shock phase) followed by a persistent stage of mitochondrial dysfunction and bioenergetic failure characterized by loss of tissue ATP, immunosuppression, sepsis, hypermetabolism, peripheral insulin resistance, overproduction of reactive oxygen species, impaired wound healing and chronic pain, among other complications. This phase may sometimes result in multiple organ failure and death of the patient. No fully effective intervention for these complications of stress and trauma has been developed to date.
Persistent dysfunction consequent to stress or trauma may be associated with changes in cellular chromatin (epigenetic remodeling). Among the best-understood types of epigenetic reprogramming include histone modifications and DNA methylation, especially in the promoter regions of genes. Epigenetic modification offers mammalian cells a powerful strategy for rapid, large-scale adjustment of transcriptional and post-transcriptional mechanisms in response to changing environmental challenges.
Epigenetic remodeling has been implicated in a variety of important disease and pathological processes as well as in aging. Among many examples: chronic kidney disease (Dwivedi R S et al, 2011); cancer (Choi J D and Lee J-S, 2013); alcohol exposure (Zachari S, 2013); chronic pain (Buchheit T et al, 2012); psychiatric disorders (Schmitt A et al, 2014); lupus (Hedrich C M and Tsokos G C, 2011); and aging (Cencioni C. et al, 2013).
Epigenetic switches may explain the underlying biology of many chronic pathophysiological processes. The so-called diseases of western civilization (chronic conditions such as arthritis, lupus, psoriasis, asthma, painful bladder syndrome, colitis, neuropathic pain, fibromyalgia and other immune-mediated diseases, osteoporosis, autism, atherosclerosis and other cardiovascular diseases, cancers and metastases of the breast, prostate and colon, metabolic syndrome-related conditions such as cardiovascular dysfunctions, diabetes, pulmonary arterial hypertension and polycystic ovary syndrome, nicotine- and other xenobiotic-related pathologies, neurodegenerative conditions such as Parkinson's and Alzheimer's, and ophthalmic diseases such as macular degeneration) are now increasingly viewed as associated with chronic relapsing-remitting inflammation cycles which, in turn, relate to epigenetically modified mechanisms. One common feature of nearly all of the emerging diseases in the Western world is the complexity and inter-relatedness of the underlying neuroimmunological and bioenergetic (mitochondrial) dysfunctions. Epigenetic narratives can satisfy the explanatory demands of nearly all of these complex conditions. New methodology and reagents for identifying the best points for mechanistic intervention in such putatively epigenetically controlled disease conditions is needed. Such methodology would provide a first step to the development of predictive diagnostics and adequately targeted interventions.
The inventor has recently shown that a central regulator of responses to metabolic and xenobiotic stress is the highly conserved Rictor-containing complex in mammalian cells known as mTORC2. Neurogenic inflammation pursuant to stress is associated with the release of neuropeptides such as substance P from nerve fibers in organ tissues. In kidney, a key early-warning organ, nephrilin (an inhibitor of Rictor complex) controls substance P levels in tissue following xenobiotic and traumatic stress, as well as downstream events such as macrophage activity, plasma IL-6 and TNF-alpha, tissue phosphorylation of p66shc, gene expression and other markers of oxidative damage and inflammation (Mascarenhas D et al [2012]; Mascarenhas D et al [2014]).
TOR (target of rapamycin) proteins are conserved Ser/Thr kinases found in diverse eukaryotes ranging from yeast to mammals. The TOR kinase is found in two biochemically and functionally distinct complexes, termed TORC1 and TORC2 (Rictor complex). Aided by the compound rapamycin, which specifically inhibits TORC1, the role of TORC1 in regulating translation and cellular growth has been extensively studied. mTORC2/Rictor complex is largely rapamycin insensitive and seems to function upstream of Rho GTPases to regulate the actin cytoskeleton (Jacinto E, et al [2004] Nat Cell Biol. 6: 1122-1128). The physiological roles of TORC2 have remained largely elusive due to the lack of pharmacological inhibitors and its genetic lethality in mammals. PRR5/PROTOR and its related family of proteins are a new class of molecules found in association to mTORC2 complex, and may be required cofactors for the function of this central regulator of neuroimmunological responses to stress. The PRR5/PROTOR gene encodes a conserved proline-rich protein predominant in kidney (Johnstone C N et al [2005] Genomics 85: 338-351). The PRR5/PROTOR class of proteins is believed to physically associate with mTORC2 and regulate aspects of growth factor signaling and apoptosis (Woo S Y et al [2007] J. Biol. Chem. 282: 25604-25612; Pearce L R et al [2007] Biochem J. 405: 513-522; Thedieck K et al [2007] PLoS ONE 2: e1217). The inventor has demonstrated the importance of a particular domain within PRR5/PROTOR comprising the sequence HESRGVTEDYLRLETLVQKVVSPYLGTYGL (SEQ ID NO:3). This sequence is conserved in human PRR5/PROTOR isoforms as well as in rat and mouse. Other obligate partners of Rictor, a central defining protein component of the mTORC2 complex, include Sin1 (also known as MIP1). Sin1 is an essential component of TORC2 but not of TORC1, and functions similarly to Rictor, the defining member of TORC2, in complex formation and kinase activity. Knockdown of Sin1 decreases Akt phosphorylation in both Drosophila and mammalian cells and diminishes Akt function in vivo. It also disrupts the interaction between Rictor and mTOR. Furthermore, Sin1 is required for TORC2 kinase activity in vitro (Yang Q et al [2006] Genes Dev. 20: 2820-2832). mTOR, SIN1 and Rictor, components of mammalian (m)TORC2/Rictor complex, are required for phosphorylation of Akt, SGK1 (serum- and glucocorticoid-induced protein kinase 1), and conventional protein kinase C (PKC). This TORC2 function is growth factor independent and conserved from yeast to mammals.
Rictor complex activity was elevated in glioma cell lines as well as in primary tumor cells as compared with normal brain tissue (Masri J et al [2007] Cancer Res. 67: 11712-11720). In these lines Rictor protein and mRNA levels were also elevated and correlated with increased kinase activity. Xenograft studies using these cell lines also supported a role for increased Rictor complex activity in tumorigenesis and enhanced tumor growth. These data suggest that mTORC2 is hyperactivated in gliomas and functions in promoting tumor cell proliferation and invasive potential. mTORC2 and its activation of downstream AGC kinases such as PKC-alpha, SGK1 and Akt have also been implicated in cancers of the prostate and breast (Guertin D A et al [2009] Cancer Cell. 15: 148-159; Sahoo S et al [2005] Eur J Cancer. 41: 2754-2759; Guo J, et al [2008] Cancer Res. 68: 8473-8481).
IRS-1 and IRS-2 are master traffic regulators in intracellular signal transduction pathways associated with growth and metabolism, playing key roles in the docking of accessory proteins to phosphorylated insulin and IGF receptors. Although similar in function, activated IRS-1 and IRS-2 proteins generate subtly different cellular outcomes, at least in part through the phosphorylation of different Akt (especially Akt 1 and Akt 2) and MAP kinase isoforms.
In diabetic humans and db/db mice the receptor for advanced glycated end products (RAGE) is activated by systemic ligands such as amphoterin, S100A9 and glycated hemoglobin (Goldin A et al [2006] Circulation 114: 597-605) and affects urinary albumin and/or NGAL (lipocalin-2). RAGE has been implicated in the development of kidney dysfunction consequent to elevated blood sugar (Tan A L et al [2007] Semin. Nephrol. 27:130-143). The inventor has recently shown that inhibition of Rictor complex and PKC reduces urinary albumin and NGAL in diabetic models (Singh B K and Mascarenhas D [2008] Am J Nephrol 28:890-899; Singh B K et al [2010] Metab Syn Relat Dis. 8(4): 1-10).
Variability within patient populations creates numerous problems for medical treatment. Without reliable means for determining which individuals will respond to a given treatment, physicians are forced to resort to trial and error. Because not all patients will respond to a given therapy, the trial and error approach means that some portion of the patients must suffer the side effects (as well as the economic costs) of a treatment that is not effective in that patient. It is therefore desirable to develop diagnostic methods and reagents to facilitate the identification of patients most likely to benefit from treatment for epigenetic reprogramming.
For some therapeutics targeted to specific locations within the body, screening to determine eligibility for the treatment can be performed. For example, the estrogen antagonist tamoxifen targets the estrogen receptor, so it is normal practice to only administer tamoxifen to those patients whose tumors express the estrogen receptor. Likewise, the anti-tumor agent trastuzumab (HERCEPTIN®) acts by binding to a cell surface molecule known as HER2/neu; patients with HER2/neu negative tumors are not normally eligible for treatment with trastuzumab. Methods for predicting whether a patient will respond to treatment with IGF-I/IGFBP-3 complex have also been disclosed (U.S. Pat. No. 5,824,467), as well as methods for creating predictive models of responsiveness to a particular treatment (U.S. Pat. No. 6,087,090).
The inventor has previously disclosed certain IGFBP-derived peptides known as “MBD” peptides (U.S. patent application publication nos. 2003/0059430, 2003/0161829, and 2003/0224990). These peptides have a number of properties, which are distinct from the IGF-binding properties of IGFBPs, that make them useful as therapeutic agents. MBD peptides are internalized some cells, and the peptides can be used as cell internalization signals to direct the uptake of molecules joined to the MBD peptides (such as proteins fused to the MBD peptide). Therapeutic peptides are provided by U.S. Pat. Nos. 7,618,816; 7,611,893; 7,662,624; and U.S. Patent Application Publication No. 2008/003,9393 A1; 2010.0152113 A1; the contents of each are hereby incorporated by reference is its entirety.
Combination treatments are increasingly being viewed as appropriate strategic options for designed interventions in complex disease conditions such as cancer, metabolic diseases, vascular diseases and neurodegenerative conditions. For example, the use of combination pills containing two different agents to treat the same condition (e.g. metformin plus a thiazolidinedione to treat diabetes, a statin plus a fibrate to treat hypercholesterolemia) is on the rise. It is therefore appropriate to envisage combination treatments that include moieties such as MBD in combination with other agents such as other peptides, antibodies, nucleic acids, chemotherapeutic agents and dietary supplements. Combinations may take the form of covalent extensions to the MBD peptide sequence, other types of conjugates, or co-administration of agents simultaneously or by staggering the treatments i.e. administration at alternating times.
The inventor has previously shown that MBD peptide-mediated delivery of bioactive molecules in vivo can be applied to disease processes such as cancer (Huq A, et al [2009] Anti-Cancer Drugs 20: 21-31) and diabetes, as described above. Nephrilin, a peptide containing the MBD scaffold, is bioactive in reducing albuminuria in diabetic mice. Nephrilin was designed to interfere with mTORC2 complex and has been shown to disrupt the association of IRS proteins with Rictor (Singh B K et al [2010] Metab Syn Relat Dis. 8(4): 1-10; U.S. Pat. No. 7,662,624). Similar approaches may be used to disrupt mTORC2 and IRS protein activity in human disease by competing the physical interaction of Rictor with obligate cofactors such as PRR5/PROTOR or Sin1/MIP1. The competing molecule may be a cell-penetrating peptide, protein, antibody or nucleic acid, or a small chemical molecule. In this work we describe in vitro assay systems that facilitate rapid screening of candidate molecules for such a purpose. Any metabolic, systemic, degenerative, or inflammatory disease process may be a candidate for interventions using such molecules.
The central role played by mTORC2 in regulating diseases of stress and aging has not been well documented. Nephrilin, an inhibitor of the binding of Rictor—the canonical component of mTORC2 complex—to its binding partners or cofactors such as PRR5/PROTOR, Sin1 and IRS proteins, is the only specific inhibitor of its class described to date. The inventor has shown that nephrilin can reverse immunological dysfunctions and disease processes relating to complications of diabetes and hypertension; acute kidney injury from rhabdomyolysis or xenotoxic stress with platinum compounds or aminoglycoside antibiotics; cancer metastasis; the neuroimmunological sequelae of burn trauma; and mortality from sepsis. These results implicate mTORC2 as a central regulator of diseases of aging. Fundamental common mechanisms suggested for the gamut of diseases of aging—the so-called diseases of western civilization—include oxidative stress [Pinton P and Rizzuto R (2008) Cell Cycle. 7(3): 304-308], loss of circadian circuitry [Uchida Y et al (2010) Biol. Pharm. Bull. 33(4) 535-544], loss of selective protein turnover mechanisms [Hussain S et al (2009) Cell Cycle 8:11, 1688-1697], and the epithelial-mesenchymal transition, EMT [Slattery C et al (2005) American Journal of Pathology, 167(2): 395-407]. The inventor has shown that biochemical signatures associated with each of these pathways can be reversed by treatment with nephrilin and has demonstrated, for the first time, that specific inhibition of mTORC2 may be the key to controlling diseases of stress and aging. Thus, therapeutic agents that disrupt binding of Rictor (the canonical component mTORC2) to its binding partners are of particular interest in the treatment of metabolic and cardiovascular diseases, especially those characterized by some underlying combination of insulin resistance, hyperglycemia, hypertension and hyperlipidemia; cancer progression and metastasis; acute kidney injury (AKI) in critical care settings, also including sepsis, systemic inflammatory conditions such as shock, post-operative stress such as after cardiopulmonary bypass or transplant, burns, pancreatitis, rhabdomyolysis, xenobiotic stresses caused by cocaine, alcohol, aminoglycoside antibiotics, antiviral compounds or platinum compounds; neurodegenerative diseases such as Parkinson's Alzheimer's, Huntington's and ALS/Lou Gehrig's disease; ototoxicities; autoimmune conditions such as lupus erythematosus and multiple sclerosis; genetic diseases such as cystinosis, Fanconi's and other conditions affecting mitochondrial respiration; pulmonary diseases, especially COPD and asthma and pulmonary arterial hypertension; migraine; ocular diseases such as cataracts and retinopathies, especially diabetic complications; and liver diseases, including chronic viral infections such as hepatitis. These disease states are now increasingly viewed as secondary to chronic inflammatory conditions that may, in turn, relate to neurogenic signaling and oxidative stress. A correlation between oxidative stress and processes of aging may explain the rising incidence of these diseases as a direct consequence of an aging population.
A key regulator of oxidative damage and aging is the adapter protein p66shc. Activation of this molecule by phosphorylation at serine 36 leads to mitochodrial translocation and increased production of free oxygen radicals. P66shc gene knockout mice live significantly longer and are protected from many of the diseases of aging listed above [Pinton P and Rizzuto R (2008) Cell Cycle. 7(3): 304-308]. The inventor has shown for the first time, that mTORC2 regulates the activation of protein kinase C beta-II (PKC-beta-II) by phosphorylation at threonine 641. PKC-beta was shown to be the activator of p66shc by phosphorylation of S36 [Pinton P, et al. (2007) Science 315: 659-663]. Nephrilin reverses both PKC-beta-II-T641 and p66shc-S36 phosphorylation events [Mascarenhas D et al, 2012].
A recently recognized histological consequence of cellular stress is the formation of microscopically visible punctate structures in or around the nuclei of stressed cells [Bart J. et al (2008) Cytometry 73A: 816-824]. The inventor has shown, in diseased hypertensive animals, the presence of such structures in kidney cells by immunohistochemical staining. The incidence of such structures is much reduced in animals treated with the mTORC2 inhibitor, nephrilin.
Epithelial cells of renal proximal tubules (PTECs) are known to be exquisitely sensitive to p66shc-mediated oxidative stress [Sun L et al (2010) Am J Physiol Renal Physiol. 299(5): F1014-F1025]. Damage to PTECs can be monitored by measuring albumin or lipocalin-2/NGAL in urine by using commercially available kits. In many of the proinflammatory disease conditions listed above, elevated levels of NGAL or albumin have been documented. This is especially true of AKI settings such as those encountered in patients with burns, hypoperfusion, pancreatitis and sepsis [Cruz D et al (2010) Intensive Care Med 36:444-451]. In AKI, moreover, a proinflammatory condition reminiscent of human systemic inflammatory states encountered in critical care settings, as enumerated above, can be generated in experimental animals by placing artificial stress on kidneys, such as in rhabdomyolysis and gentamycin models [Zager R et al (2006) Am J Physiol Renal Physiol 291:F546-F556]. The inventor has shown, for the first time, that this type of proinflammatory state can be successfully treated by an inhibitor of Rictor complex, nephrilin.
Activated NADPH oxidases (Nox) have been implicated in the generation of reactive oxygen species, thus playing a central role in oxidative stress in the tissues. A key player in the activation of Nox is Rac1/2, a central player in cellular GTP metabolism. Activated Rac1 (phosphorylated Ser71) is a subunit of active Nox. The inventor has shown that Rac1, as well as several molecules that serve as accessories to its activity (PKC, Prex1, p66shc, Pitx2, ERK1/2) are up-regulated in the tissues of burned animals. Treatment with nephrilin can reduce activation of Rac1-S71 as well as accessory molecules (activated PKCs, activated p66shc, etc.). These pathways are known to be integral to oxidative stress as well as pain in the CNS. Nephrilin treatment may abate neuropathies, migraine and other pain-related molecules and pathways in the CNS.
A key finding of the inventor that relates to the epigenetic memory caused by early chromatin-modifying events following severe burn trauma, is that treatment of burned animals beginning at 2 hours after burn and continuing until day 7 is more efficacious than treatment beginning at day 8 and continuing to Day 14. Evaluation of animals in both cases was done at Day 14. Differences were found on glycemic control, splenomegaly (anemia), oxidative metabolism, kidney function and several clinically relevant markers of metabolism and neuroimmune function, including changes in the CNS. These results support the narrative of epigenetic remodeling described above. Basal gene expression of key activators of oxidative stress such as PKC-beta2, Prex1, Bik and Pitx2 were stably modified in tissues of burned animals, as well as activation of downstream targets such as phosphorylated Rac1-Ser71 and phosphorylated p66shc-Ser36. These activations via phosphorylation were better controlled by nephrilin treatment on days 1-7 versus 8-14.
Burned animals show impaired glucose tolerance and elevated serum insulin levels after glucose challenge. The inventor has shown that these markers of dysregulated glycemic control are normalized by treatment with nephrilin on days 1-7. Nephrilin has also been shown to normalize impaired levels of insulin C-peptide in serum.
Burned animals show elevated levels of 8-isoprostane and 8-OHDG in urine at 14 days. The inventor has shown that nephrilin treatment dramatically reduces these markers of oxidative damage in the urine.
A well-known consequence of critical illness in general and burn injury in particular is persistent anemia secondary to impaired iron metabolism and erythropoiesis. These deficits lead to pronounced splenomegaly in burned subjects. The inventor has shown, in burned rats, that treatment with nephrilin can substantially reduce splenomegay in burned animals.
All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.